EP0354819B1 - Novel peptide and anti-dementia agent - Google Patents

Novel peptide and anti-dementia agent Download PDF

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Publication number
EP0354819B1
EP0354819B1 EP89308221A EP89308221A EP0354819B1 EP 0354819 B1 EP0354819 B1 EP 0354819B1 EP 89308221 A EP89308221 A EP 89308221A EP 89308221 A EP89308221 A EP 89308221A EP 0354819 B1 EP0354819 B1 EP 0354819B1
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EP
European Patent Office
Prior art keywords
formula
alkyl group
arg
pro
peptide
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EP89308221A
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German (de)
French (fr)
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EP0354819A3 (en
EP0354819A2 (en
Inventor
Mitsuo Masaki
Norihisa Miyake
Masaki Uehara
Kenji Hirate
Yoshikazu C/O Fujirebio Kabushiki Kaisha Isowa
Yoshiaki C/O Fujirebio Kabushiki Kaisha Satoh
Yoshiharu Nakashima
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Fujirebio Inc
Nippon Chemiphar Co Ltd
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Fujirebio Inc
Nippon Chemiphar Co Ltd
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Priority claimed from JP63201358A external-priority patent/JP2654673B2/en
Priority claimed from JP63201359A external-priority patent/JP2542241B2/en
Application filed by Fujirebio Inc, Nippon Chemiphar Co Ltd filed Critical Fujirebio Inc
Priority to AT89308221T priority Critical patent/ATE104314T1/en
Publication of EP0354819A2 publication Critical patent/EP0354819A2/en
Publication of EP0354819A3 publication Critical patent/EP0354819A3/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/16—Oxytocins; Vasopressins; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel peptides having a nootropic effect and an anti-dementia agent containing the same.
  • Vasopressin has been previously known as a compound having a nootropic effect, i.e., intelligence developing effect. Recently, it has been reported that a peptide seemingly corresponding to a vasopressin fragment, for example, one having the following formula: has such a nootropic effect as that of vasopressin in Science, 221 , pp.1310-1312 (1983).
  • Japanese Patent Provisional Publication No.59(1984)-93036 describes that a peptide having the formula: also has a nootropic effect.
  • the present invention provides a peptide having one of the following formulae (I), (II), (III) and (IV): pGlu-Asn-Cys-Pro-Arg-Gly (II) Asn-Cys-Pro-Arg (IV) and functional derivatives thereof selected from derivatives of N-acyl groups of formula -NHCOR where R is an alkyl group having 1-5 carbon atoms; amide, monoalkyl- or dialkyl-substituted amides of formula -CONH2, -CONHR or -CONR2 where R is an alkyl group having from 1 to 6 atoms; and ester groups of formula -COOR1 where R1 is an alkyl group having from 1-18 carbon atoms.
  • the novel peptides of the invention can be in the form of their pharmaceutically acceptable salts.
  • the peptides of the invention have one of the aforementioned formulae (I), (II), (III) and (IV) and may be in the form of their functional derivatives.
  • acid addition salts and basic salts such as alkali metal salts and ammonium salts can be mentioned.
  • acid adition salts include salts of inorganic acids (e.g., hydrochloric acid, sulfuric acid and phosphoric acid) or of organic acids (e.g., acetic acid, propionic acid, citric acid, tartaric acid, malic acid, oxalic acid and methanesulfonic acid).
  • basic salts include sodium salt, potassium salt, and triethylamine salt.
  • the peptides are described by abbreviations commonly used in the field of chemistry, or abbreviations recommended by the IUPAC-IUB Commission on Biochemical Nomenclature .
  • the following symbols are used in the specification.
  • the amino acids should be construed to be of the L-type, unless specific description with respect to optical configuration is given.
  • Examples of the methods for formation of the peptide bonds include azide method, acid chloride method, symmetrical anhydride method, mixed anhydride method, N,N′-dicyclohexylcarbodiimide method, N,N′-dicyclohexylcarbodiimido-additive method, activated ester method, carbonyldiimidazole method, oxidation-reduction method, and the one employing a Woodward reagent K.
  • the cystine moiety which is an amino acid forming the peptide of the invention can be formed by employing a cystine derivative or by converting a cysteine moiety of the peptide chain into a cystine moiety after the formation of the peptide chain by the conventional method.
  • carboxyl group, amino group, guanidino group and mercapto group and the like which do not participate in the reaction can be protected, and those which participate in the coupling reaction can be activated, both by the methods well known in the art.
  • protecting groups for the carboxyl group include ester-forming groups such as methyl, ethyl, benzyl, p-nitrobenzyl, t-butyl and cyclohexyl.
  • Examples of the protecting groups for the amino group include benzyloxycarbonyl, t-butoxycarbonyl, isobornyloxycarbonyl, and 9-fluorenylmethyloxycarbonyl.
  • Examples of the protecting groups for the guanidino group include nitro, benzyloxycarbonyl, tosyl, p-methoxy-benzenesulfonyl, and mesitylensulfonyl.
  • Examples of the protecting groups for the mercapto group include trityl, acetamidomethyl, benzyl, p-methoxy-benzyl; and 3-nitro-2-pyridinesulfenyl.
  • Examples of the activation of carboxyl group include symmetrical anhydride, mixed anhydride, azide and active ester (ester with alcohol e.g., pentachlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboxyimide, N-hydroxyphthalimide, and 1-hydroxybenzotriazol).
  • An example of the activation of amino group is phosphite-amide.
  • the reaction is generaly carried out in a solvent such as chloroform, dichloromethane, ethyl acetate, N,N-dimethylformamide, dimethylsulfoxide, pyridine, dioxane, tetrahydrofuran, water, methanol and mixture of these solvents.
  • a solvent such as chloroform, dichloromethane, ethyl acetate, N,N-dimethylformamide, dimethylsulfoxide, pyridine, dioxane, tetrahydrofuran, water, methanol and mixture of these solvents.
  • the reaction temperature may be in the range of approx. -30°C to 50°C, which is generally employed for the reaction.
  • condition for removing the protecting group of the peptide of the invention may differ depending on the kind of the blocking group, but it should be the one rich is able to release the blocking group without giving any influence to the peptide bonding.
  • the protecting group can be removed by acid treatment, for example, treatment with hydrogen chloride, hydrogen bromide, hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, and mixture of these acids. Further, the reduction with sodium metal in liquid ammonia or catalytic hydrogenolysis over palladium-carbon can be employed. On the reaction for removing the protecting group by the above acid treatment, addition of cation scavenger such as anisole, phenol and thioanisole is advantageous.
  • the prepared peptide of the present invention can be obtained by the conventional process for purification of peptides, for example, extraction, partition, reprecipitation, recrystallization or column chromatography.
  • peptides of the present invention can be converted into their functional derivatives or their pharmaceutically acceptable salts as described above by the conventional manner.
  • the peptides of the present invention show a strong nootropic effect in passive avoidance tests using rats as described hereinafter.
  • the peptide of the present invention is effective for the following diseases and can be used for prevention or treatment thereof: senile dementia (Alzheimer's dementia), cerebrovascular dementia, and dementia based on Alzheimer's disease, Pick's disease, Huntington's disease, Creutzfeldt-Jakob disease, Parkinson's disease, cerebellar myelic denatured disease.
  • senile dementia Alzheimer's dementia
  • cerebrovascular dementia dementia based on Alzheimer's disease
  • Pick's disease Huntington's disease
  • Creutzfeldt-Jakob disease Creutzfeldt-Jakob disease
  • Parkinson's disease cerebellar myelic denatured disease.
  • the peptides of the present invention have an extremely low toxicity, and cause no death even by administration at extremely higher dose than their effective dose.
  • the peptides of the present invention may be in its inner salt form, its fanctional derivatives, or salts thereof. No matter their forms is, the dose as amount of the peptides of the formula (I) are preferably in the range of 1 ng/day to 1 mg/day per 1 kg of a patient. In the case of parenteral administration (excluding rectal administration), the dose preferably is in the range of 10 ng/kg to 100 ⁇ g/kg per day. In the case of oral administration and rectal administration, it is preferred that the dose should be 10 to 100 times to that of the parenteral administration (excluding rectal administration).
  • the peptides of the present invention are mainly administered parenterally (e.g., intravenous or hypodermic injection, intracerebroventricular or intraspinal administration, nasal administration, and rectal administration). They can be also administered orally depending on the case.
  • the peptide of the present invention can be incorporated into a pharmaceutical composition in the form of injection liquid, suppository, powder, collunarium, granule and tablets.
  • the peptides of the invention can be preserved as physiological saline solutions or can be freeze-dried in an ample after addition of mannitol or sorbitol and are melted when they are used for administration.
  • the eluants used for a thin-layer chromatography were as follows.
  • TLC Plate Silica Gel 60F254 by Merck Co., Ltd. was used.
  • N,N-dimethylformamide DMF
  • N,N-dimethylformamide DMF
  • N,N′-dicyclohexylurea was removed from the mixture by filtration, and the filtrate was treated to distill off DMF.
  • the solvent was distilled off, and the residue was crystallized from methanol-ether to yield the desired compound by filtration.
  • N,N′-dicyclohexylurea was removed by filtration, and the filtrate was treated to distill DMF.
  • the residue was dissolved in 2-butanol-dichloromethane (5:1 v/v), the resulting solution was successively washed with a saturated aqueous sodium hydrogencarbonate solution, dilute hydrochloric acid solution and a saturated aqueous saturated with sodium chloride, an aqueous sodium chloride solution, and dried over anhydrous sodium sulfate.
  • the solvent was distilled off, and the residue was crystallized by addition of ether and collected by filtration to yield the desired compound.
  • reaction mixture was filtered to remove N,N′-dicyclohexylurea, and the filtrate was treated to distill off DMF.
  • the solvent was distilled off, and the residue was crystallized by addition of ether and collected by filtration to yield the desired compound.
  • the solvent was distilled off.
  • the residue was crystallized by addition of ether, and the crystals were collected by filtration to give the desired compound.
  • the solvent was distilled off, and the residue was purified by a silica gel column chromatography using chloroform-methanol, and then crystallized by addition of ether. The precipitated crystals were collected by filtration to give the desired compound.
  • the residue was dissolved in 0.05 % trifluoroacetic acid, and the solution was purified using a high performance liquid chromatography at 15 ml/min. (flow rate), and 0 to 10 % B) 20 min. linear gradient (mobile phase).
  • the resulting solution was treated with Dowex 1x2 (acetate type), and then freeze-dried to give the desired compound.
  • Example 1 The procedure of (1) in Example 1 was repeated using 10 g of Z-Arg(Mbs)-OH dicyclohexylamine salt, 1.7 g of H-Gly-NH2 hydrochloride, 1.7 ml of N-methylmorpholine, 2g of 1-hydroxybenzotriazole and 3.4 g of N,N′-dicyclohexylcarbodiimide to give the desired compound.
  • Example 2 The procedure of (2) in Example 1 was repeated using 20.8 g of Z-Arg(Mbs)-Gly-NH2, 12.1 g of Boc-Pro-OSu and 4.3 ml of N-methylmorpholine to give the desired compound.
  • the solvent was distilled off, and ether was added to the resulting residue for crystallization.
  • the crystals were collected by filtration to yield the desired compound.
  • the solvent was distilled off, and the residue was purified by silica gel chromatography using chloroform-methanol, and then crystallized by addition of ether and collected by filtration to give the desired compound.
  • the residue was dissolved in 0.05 % trifluoroacetica cid, the solution was purified using a high-performance liquid chromatography at 15 ml/min. (flow rate), 2 to 12 % B) 20 min. linear gradient (mobile phase). The resulting solution was treated with Dowex 1x2 (acetate type) treatment and freeze-dried to give the desired compound.
  • Example 2 The procedure of (3) in Example 1 was repeated using 2.5 g of Boc-D-Pro-Arg(Mbs)-Gly-NH2, 1.3 g of Boc-Cys(Acm)-OH, 0.73 g of 1-hydroxybenzotriazole, 0.94 g of N,N′-dicyclohexylcarbodiimide and 1 ml of N-methylmorpholine to give the desired compound.
  • Example 2 The procedure of (4) in Example 1 was repeated using 2.0 g of Boc-Cys(Acm)-D-Pro-Arg(Mbs)-Gly-NH2, 2.0 g of Z-pGlu-Asn-OH, 0.5 g of 1-hydroxybenzotriazole, 0.58 g of N,N′-dicyclohexylcarbodiimide and 0.5 ml of N-methylmorpholine to give the desired compound.
  • Example 2 The procedure of (5) in Example 1 was repeated using 1.0 g of Z-pGlu-Asn-Cys(Acm)-D-Pro-Arg(Mbs)-Gly-NH2 and 0.15 ml of Cl-Scm to give the desired compound.
  • THF was distilled off, and the residue was dissolved in ethyl acetate.
  • the solution was successively washed with an aqueous dilute hydrochloric acid, a saturated aqueous sodium hydrogencarbonate solution and water, and dried over anhydrous sodium sulfate.
  • the solvent was distilled off to give the desired compound as an oil.
  • the solvent was distilled off, and the residue was purified by shilica gel chromatography using chloroformmethanol to yield the desired compound as an oil.
  • the effect of peptides of the invention on memory consolidation was evaluated by conducting one-trial passive avoidance experiment using male Wistar rats in accordance with the method described by Burbach et al., Science , vol. 221, pp. 1310-1312, 1983.
  • the apparatus was composed of an illuminated room and a dark room, and their floors were made of stainless-steel grid.
  • the rats placed in the illuminated room could freely enter the dark room.
  • the rats received an electro-shock.
  • Retention of passive avoidance behavior to the electro-shock was determined by the measurement of a response latent period, i.e. period required for the rat experienced the electro-shock to reenter the dark room from the time at which the rat was placed in the illuminated room after predetermined intervals.
  • the rats were treated with the peptides of the invention obtained in the aforementioned Example 1 to 4 or a physiological saline solution by means of subcutaneous injection immediately after receiving the electro-shock (0.25 mA). Then 24 hours later, the retention of the memory of the elctro-shock was tested.
  • the rats administered with the physiological saline solution alone as a control group generally showed response latent period of approx. 50 seconds.
  • the rats received an electro-shock (0.5 mA) after one hr. from the administration of the peptides of the invention or a physiological saline solution. Immediately after receiving the electro-shock, the rats were treated with 2.7 to 3.0 mg/kg of cycloheximide or the saline solution by subcutaneous injection. At 48 hours after the administration was made, memory retentions of the rats were tested.
  • the rats administered with only the physiological saline solution showed the response latent period of approx. 300 seconds, and those rats of control group administered with a physiological saline and treated with cycloheximide alone showed the response latent period of approx. 50 seconds, which revealed retrograde amnesia.
  • the average response latent period of rats administered with each peptide of the invention and treated with cycloheximide were compared with that of the control group. Six to eight rats were used for each group to be tested. The response latent period was measured up to a maximum of 600 seconds.
  • the peptides of the invention had the same effects as the known peptides at a dose of 1/10 to 1/100 to that of the known peptides and showed superior effect on the facilitation of memory consolidation as well as effect on improving retrograde amnesia.
  • the structures of the peptides of the invention have similar structures to those of the known peptides, they are slightly different from those of the known peptides which hold the significant influence on memory consolidation effect of a peptide.
  • the fact that these slight defferences made the peptides of the invention produce remarkable effect indicates that it is impossible to make an estimation on memory consolidation effect of a peptide from its structure, and that the peptides of the invention have the uniqueness on their effects.
  • Example 1 Although the peptide obtained in Example 1 possesses D-Arg in place of L-Arg of the peptide of Comparison Compound 1 and the rest of the structure of each peptide is identical, the peptide obtained in Example 1 showed the same effect to that of Comparison Compound 1 with only 1/10 of dose, which testifies the fact the the peptide obtained in Example 1 has much seperior effect to the known peptide.
  • Example 2 To 100 ml of a distilled water for injection were added 0.1 mg of the peptide obtained in Example 1 and 0.9 g of sodium chloride to prepare an aqueous solution whose pH was adjusted to 6.0 to 8.0 with sodium hydroxide. The solution was filtered under sterile condition, and the filtrate was filled up into 1 ml ampul. The ampul was fused to seal under sterile condition by heating to prepare an agent for injection.
  • Example 2 To 100 ml of a distilled water for injection were added 5 mg of the peptide obtained in Example 1 and 5 g of D-mannitol to prepare an aqueous solution of which pH was adjusted to 6.0 to 8.0 with a phosphate buffer. The solution was filtered under sterile condition and the filtrate was divided into a plurality of 1 ml vials. The divided portions were freeze-dried to prepare a freeze-dried agent for injection.
  • Example 2 To 100 ml of a physiological saline solution was added 10 mg of the peptide obtained in Example 1. The pH of the mixture was adjusted to 3.0 to 6.0 with a citric acid buffer to prepare a collinarium which contains 50 ⁇ g of the peptide of the invention in a dose of 0.5 ml.

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Description

    BACKGROUND OF THE INVENTION Field of the invention
  • The present invention relates to novel peptides having a nootropic effect and an anti-dementia agent containing the same.
    • Description of prior art
  • Vasopressin has been previously known as a compound having a nootropic effect, i.e., intelligence developing effect. Recently, it has been reported that a peptide seemingly corresponding to a vasopressin fragment, for example, one having the following formula:
    Figure imgb0001
    has such a nootropic effect as that of vasopressin in Science, 221, pp.1310-1312 (1983).
  • Further, Japanese Patent Provisional Publication No.59(1984)-93036 describes that a peptide having the formula:
    Figure imgb0002
    also has a nootropic effect.
    • SUMMARY OF INVENTION
  • It is an object of the present invention to provide new peptides having a nootropic effect which is superior to the known vasopressin as well as to the known peptides corresponding to vasopressin fragments.
  • The present invention provides a peptide having one of the following formulae (I), (II), (III) and (IV):
    Figure imgb0003


            pGlu-Asn-Cys-Pro-Arg-Gly   (II)

    Figure imgb0004


            Asn-Cys-Pro-Arg   (IV)



    and functional derivatives thereof selected from derivatives of N-acyl groups of formula -NHCOR where R is an alkyl group having 1-5 carbon atoms; amide, monoalkyl- or dialkyl-substituted amides of formula -CONH₂, -CONHR or -CONR₂ where R is an alkyl group having from 1 to 6 atoms; and ester groups of formula -COOR¹ where R¹ is an alkyl group having from 1-18 carbon atoms. The novel peptides of the invention can be in the form of their pharmaceutically acceptable salts.
  • The above-mentioned peptides, their functional derivatives, and their pharmaceutically acceptable salts show a prominent nootropic effect in passive avoidance tests using rats, and are prominently effective as the active component of pharmaceutical agents for prevention or treatment of senile dementia (Alzheimer's dementia), cerebrovascular dementia and other dementia diseases.
    • DETAILED DESCRIPTION OF THE INVENTION
  • The peptides of the invention have one of the aforementioned formulae (I), (II), (III) and (IV) and may be in the form of their functional derivatives.
  • The functional derivatives of the peptides of the formulae (I), (II), (III) and (IV) are selected from the following derivatives:
    • a) N-acyl derivatives having N-acyl group(s) at the functional group(s); N-acyl group is derived from an aliphatic carboxylic acid having 1 to 6 carbon atoms, preferably one derived from acetic acid; the N-acyl group can be expressed by -NHCOR (wherein R is an alkyl group having 1-5 carbon atoms),
    • b) derivatives having, at the functional group(s), groups in the form of amides, or monoalkyl or dialkyl substituted-amides having alkyl chain(s) of 1 to 6 carbon atoms; which can be expressed by -CONH₂, -CONHR, and -CONR₂ (wherein R is an alkyl group having 1-6 carbon atoms), and
    • c) derivatives having, at the functional group(s) in the form of esters derived from alcohol having 1 to 18 carbon atoms, preferably those derived from an aliphatic alcohol having 1 to 6 carbon atoms; which can be expressed by -COOR (wherein R is an alkyl group having carbon 1 - 18 atoms, preferably 1 - 6 carbon atoms).
  • As the examples of pharmaceutically acceptable salts of the peptides or their derivatives, acid addition salts and basic salts such as alkali metal salts and ammonium salts can be mentioned. Examples of such acid adition salts include salts of inorganic acids (e.g., hydrochloric acid, sulfuric acid and phosphoric acid) or of organic acids (e.g., acetic acid, propionic acid, citric acid, tartaric acid, malic acid, oxalic acid and methanesulfonic acid). Examples of basic salts include sodium salt, potassium salt, and triethylamine salt.
  • In the specification, the peptides are described by abbreviations commonly used in the field of chemistry, or abbreviations recommended by the IUPAC-IUB Commission on Biochemical Nomenclature . For example, the following symbols are used in the specification. The amino acids should be construed to be of the L-type, unless specific description with respect to optical configuration is given.
    • Asn :
    asparagine
    Arg :
    arginine
    Cys :
    cysteine
    Gly :
    glycine
    pGlu :
    pyroglutamic acid
    Pro :
    proline
    Boc :
    t-butoxycarbonyl
    Z :
    benzyloxycarbonyl
    Mbs :
    p-methoxybenzenesulfonyl
    MBzl :
    p-methoxybenzyl
    Acm :
    Acetamidomethyl
    Scm :
    S-carbomethoxysulfenyl
    OBzl :
    benzyl ester
    OSu :
    N-hydroxysuccinimide ester
       The compounds of the present invention can be prepared by the methods conventionally employed in peptide chemistry. For example, they can be prepared by those processes described in Schröder and Lübke, The Peptides, Vol 1, Academic Press, New York, 1965. and Nobuo Izumiya et al., Fundamental and Experiment of Peptide Synthesis, Maruzen, Tokyo, 1985, and can be prepared by either the solution synthesis or the solid phase synthesis.
  • Examples of the methods for formation of the peptide bonds include azide method, acid chloride method, symmetrical anhydride method, mixed anhydride method, N,N′-dicyclohexylcarbodiimide method, N,N′-dicyclohexylcarbodiimido-additive method, activated ester method, carbonyldiimidazole method, oxidation-reduction method, and the one employing a Woodward reagent K.
  • In the synthesis of peptide, the cystine moiety which is an amino acid forming the peptide of the invention can be formed by employing a cystine derivative or by converting a cysteine moiety of the peptide chain into a cystine moiety after the formation of the peptide chain by the conventional method.
  • Before carrying out the coupling reaction, carboxyl group, amino group, guanidino group and mercapto group and the like which do not participate in the reaction can be protected, and those which participate in the coupling reaction can be activated, both by the methods well known in the art.
  • Examples of the protecting groups for the carboxyl group include ester-forming groups such as methyl, ethyl, benzyl, p-nitrobenzyl, t-butyl and cyclohexyl.
  • Examples of the protecting groups for the amino group include benzyloxycarbonyl, t-butoxycarbonyl, isobornyloxycarbonyl, and 9-fluorenylmethyloxycarbonyl.
  • Examples of the protecting groups for the guanidino group include nitro, benzyloxycarbonyl, tosyl, p-methoxy-benzenesulfonyl, and mesitylensulfonyl.
  • Examples of the protecting groups for the mercapto group include trityl, acetamidomethyl, benzyl, p-methoxy-benzyl; and 3-nitro-2-pyridinesulfenyl.
  • Examples of the activation of carboxyl group include symmetrical anhydride, mixed anhydride, azide and active ester (ester with alcohol e.g., pentachlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboxyimide, N-hydroxyphthalimide, and 1-hydroxybenzotriazol). An example of the activation of amino group is phosphite-amide.
  • The reaction is generaly carried out in a solvent such as chloroform, dichloromethane, ethyl acetate, N,N-dimethylformamide, dimethylsulfoxide, pyridine, dioxane, tetrahydrofuran, water, methanol and mixture of these solvents.
  • The reaction temperature may be in the range of approx. -30°C to 50°C, which is generally employed for the reaction.
  • The condition for removing the protecting group of the peptide of the invention may differ depending on the kind of the blocking group, but it should be the one rich is able to release the blocking group without giving any influence to the peptide bonding.
  • The protecting group can be removed by acid treatment, for example, treatment with hydrogen chloride, hydrogen bromide, hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, and mixture of these acids. Further, the reduction with sodium metal in liquid ammonia or catalytic hydrogenolysis over palladium-carbon can be employed. On the reaction for removing the protecting group by the above acid treatment, addition of cation scavenger such as anisole, phenol and thioanisole is advantageous.
  • After the reaction is complete, the prepared peptide of the present invention can be obtained by the conventional process for purification of peptides, for example, extraction, partition, reprecipitation, recrystallization or column chromatography.
  • Further, the peptides of the present invention can be converted into their functional derivatives or their pharmaceutically acceptable salts as described above by the conventional manner.
  • The peptides of the present invention show a strong nootropic effect in passive avoidance tests using rats as described hereinafter.
  • The peptide of the present invention is effective for the following diseases and can be used for prevention or treatment thereof: senile dementia (Alzheimer's dementia), cerebrovascular dementia, and dementia based on Alzheimer's disease, Pick's disease, Huntington's disease, Creutzfeldt-Jakob disease, Parkinson's disease, cerebellar myelic denatured disease.
  • The peptides of the present invention have an extremely low toxicity, and cause no death even by administration at extremely higher dose than their effective dose.
  • The peptides of the present invention may be in its inner salt form, its fanctional derivatives, or salts thereof. No matter their forms is, the dose as amount of the peptides of the formula (I) are preferably in the range of 1 ng/day to 1 mg/day per 1 kg of a patient. In the case of parenteral administration (excluding rectal administration), the dose preferably is in the range of 10 ng/kg to 100 µg/kg per day. In the case of oral administration and rectal administration, it is preferred that the dose should be 10 to 100 times to that of the parenteral administration (excluding rectal administration). The peptides of the present invention are mainly administered parenterally (e.g., intravenous or hypodermic injection, intracerebroventricular or intraspinal administration, nasal administration, and rectal administration). They can be also administered orally depending on the case.
  • The peptide of the present invention can be incorporated into a pharmaceutical composition in the form of injection liquid, suppository, powder, collunarium, granule and tablets. The peptides of the invention can be preserved as physiological saline solutions or can be freeze-dried in an ample after addition of mannitol or sorbitol and are melted when they are used for administration.
  • Examples of the present invention are set forth hereinafter.
  • In each example, the eluants used for a thin-layer chromatography (TLC) were as follows. As for the solid phase, TLC Plate Silica Gel 60F₂₅₄ by Merck Co., Ltd. was used.
    • Rf¹:
    chloroform-methanol-acetic acid-water (80:20:2.5:5) lower layer
    Rf²:
    chloroform-methanol-water (70:30:5)
    Rf³:
    n-butanol-acetic acid-water (2:1:1)
       Further, purification by a high-performance liquid chromatograpy was carried out using the following materials:
    Column:
    µBondapak C₁₈ 1.9 x 15 cm
    Mobile phase:
    A) 0.05% trifluoroacetic acid (TFA)
    B) acetonitrile
    Example 1
  • Figure imgb0005
    • (1) Z-D-Arg(Mbs)-Gly-NH₂
  • In a mixture of 500 ml of ethyl acetate and 200 ml of 5 % aqueous citric acid solution was dissolved 30 g of Z-D-Arg(Mbs)-OH dicyclohexylamine salt under stirring. Ethyl acetate portion was then washed with water, and dried over anhydrous sodium sulfate.
  • After the solvent was distilled off, the resulting residue was dissolved in 300 ml of N,N-dimethylformamide (DMF). To the solution were successively added under chilling with ice 5 g of H-Gly-NH₂ hydrochloride, 5 ml of N-methylmorpholin, 8 g of 1-hydroxybenzotriazole and 9.8 g of N,N′-dicyclohexylcarbodiimide. After the reaction mixture was stirred for 18 hours at room temperature, N,N′-dicyclohexylurea was removed from the mixture by filtration, and the filtrate was treated to distill off DMF.
  • The resulting residue was dissolved in 2-butanoldichloromethane (5:1 v/v), and the resulting solution was washed successively with a saturated aqueous sodium hydrogencarbonate solution, dilute hydrochloric acid saturated with sodium chloride and a saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate.
  • The solvent was distilled off, and the residue was crystallized from methanol-ether to yield the desired compound by filtration.
    • Yield :
    14.6 g
    M.P. :
    194 - 196°C
    Rf¹ :
    0.24, Rf² : 0.52
    [α]D :
    -2.9° (c=0.5, DMF)
    (2) Boc-Pro-D-Arg(Mbs)-Gly-NH₂
  • To 200 ml of 80 % acetic acid was added 10.7 g of Z-D-Arg(Mbs)-Gly-NH₂. The mixture was stirred for 6 hours in a stream of hydrogen in the presence of 10 % palladium-carbon.
  • After palladium-carbon was removed by filtration, the solvent was distilled off from the filtrate. The residue was dried under reduced pressure and dissolved in 100 ml of DMF. To the resulting solution were added 3 ml of N-methylmorpholine and 6.2 g of Boc-Pro-OSu, and the mixture was stirred for 18 hours at room temperature.
  • After DMF was distilled off, the resulting residue was dissolved in 2-butanol-dichloromethane (5:1 v/v). The resulting solution was then washed successively with a saturated aqueous sodium hydrogencarbonate solution, dilute hydrochloric acid saturated with sodium chloride and a saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate.
  • After the solvent was distilled off, the residue was crystallized from ether and collected by filtration to give the desired compound.
    • Yield :
    11.9 g
    M.P. :
    108 - 111°C
    Rf¹ :
    0.32 Rf² : 0.56
    [α]D :
    -6.9° (c=0.5, DMF)
    (3) Boc-Cys(Acm)-Pro-D-Arg(Mbs)-Gly-NH₂
  • To a mixture of 100 ml of tetrahydrofuran (THF) and 100 ml of 4N HCl-ethyl acetate was added 9 g of Boc-Pro-D-Arg(Mbs)-Gly-NH₂. The mixture was allowed to stand for 30 minutes at room temperature, and then treated to distill off the solvent.
  • The residue was dried under reduced pressure and was dissolved in 100 ml of DMF. To the resulting solution were successively added under chilling with ice 3.3 ml of N-methylmorpholin, 4.8 g of Boc-Cys(Acm)-OH, 2.4 g of 1-hydroxybenzotriazole and 3.4 g of N,N′-dicyclohexylcarbodiimide. The mixture was then stirred for 18 hours at room temperature.
  • N,N′-dicyclohexylurea was removed by filtration, and the filtrate was treated to distill DMF. The residue was dissolved in 2-butanol-dichloromethane (5:1 v/v), the resulting solution was successively washed with a saturated aqueous sodium hydrogencarbonate solution, dilute hydrochloric acid solution and a saturated aqueous saturated with sodium chloride, an aqueous sodium chloride solution, and dried over anhydrous sodium sulfate.
  • The solvent was distilled off, and the residue was crystallized by addition of ether and collected by filtration to yield the desired compound.
    • Yield :
    9.8 g
    M.P. :
    88 - 90°C
    Rf¹ :
    0.21 Rf² : 0.52
    [α]D :
    -17.6° (c=0.5, DMF)
    (4) Z-pGlu-Asn-Cys(Acm)-Pro-D-Arg(Mbs)-Gly-NH₂
  • To 20 ml of 2N HCl-acetic acid was added 6.3 g of Boc-Cys(Acm)-Pro-D-Arg(Mbs)-Gly-NH₂. After the mixture was allowed to stand for 30 minutes at room temperature, the solvent was distilled off.
  • The residue was dried under reduced pressure, and dissolved in 100 ml of DMF. To the resulting solution were successively added under chilling with ice 1 ml of N-methylmorpholin, 3.1 g of Z-pGlu-Asn-OH, 1.3 g of 1-hydroxybenzotriazole, and 1.8 g of N,N′-dicyclohexylcarbodiimide.
  • Having been stirred for 40 hours at room temperature, the reaction mixture was filtered to remove N,N′-dicyclohexylurea, and the filtrate was treated to distill off DMF.
  • In 2-butanol-dichloromethane (5:1 v/v) was dissolved the resulting residue, and the resulting solution was washed successively with a saturated aqueous sodium hydrogencarbonate solution, dilute hydrochloric acid saturated with sodium chloride and a saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate.
  • The solvent was distilled off, and the residue was crystallized by addition of ether and collected by filtration to yield the desired compound.
    • Yield :
    6.0 g
    M.P. :
    161 - 166°C
    Rf¹ :
    0.05 Rf² : 0.31
    [α]D :
    -35.0° (c=0.5, DMF)
    (5) Z-pGlu-Asn-Cys(Scm)-Pro-D-Arg(Mbs)-Gly-NH₂
  • To a solution of 1.0 g of Z-pGlu-Asn-Cys(Acm)-Pro-D-Arg(Mbs)-Gly-NH₂ in 50ml of dichloromethane-methanol (1:1 v/v) was added under chilling with ice 0.15 ml of carbomethoxysulfenyl chloride (Cl-Scm), and the mixture was stirred for 1 hour.
  • The solvent was distilled off. The residue was crystallized by addition of ether, and the crystals were collected by filtration to give the desired compound.
    • Yield :
    1.0 g
    M.P. :
    176 - 180°C
    Rf¹ :
    0.11 Rf² : 0.42
    [α]D :
    -54.3° (c=0.5, DMF)
    Figure imgb0006
       To a solution of 1.0 g of Z-pGlu-Asn-Cys(Scm)-Pro-D-Arg(Mbs)-Gly-NH₂ in 20 ml of DMF was added 0.38 g of cysteine hydrochloride. The mixture was stirred for 1 hour at room temperature.
  • The solvent was distilled off, and the residue was purified by a silica gel column chromatography using chloroform-methanol, and then crystallized by addition of ether. The precipitated crystals were collected by filtration to give the desired compound.
    • Yield :
    0.68 g
    M.P. :
    162 - 166°C
    Rf² :
    0.05
    [α]D :
    -37.9° (c=0.5, DMF)
    Figure imgb0007
       To a mixture of 4ml of methanesulfonic acid and 0.4 ml of anisole was added 420 mg of
    Figure imgb0008
    and the resluting mixture was stirred for 1.5 hours at room temperature. To the reaction mixture was added eter, and the supernatant portion of the mixture was removed. The precipitate was dissolved in water, the resulting solution was then subjected to Dowex 1x2 (acetate type) treatment, and the water was distilled off.
  • The residue was dissolved in 0.05 % trifluoroacetic acid, and the solution was purified using a high performance liquid chromatography at 15 ml/min. (flow rate), and 0 to 10 % B) 20 min. linear gradient (mobile phase). The resulting solution was treated with Dowex 1x2 (acetate type), and then freeze-dried to give the desired compound.
    • Yield :
    80 mg
    Rf³ :
    0.07
    [α]D :
    -129.1° (c=0.6, water)
    Example 2
  •    pGlu-Asn-Cys-Pro-Arg-Gly-NH₂ acetate
    • (1) Z-Arg(Mbs)-Gly-NH₂
  • The procedure of (1) in Example 1 was repeated using 10 g of Z-Arg(Mbs)-OH dicyclohexylamine salt, 1.7 g of H-Gly-NH₂ hydrochloride, 1.7 ml of N-methylmorpholine, 2g of 1-hydroxybenzotriazole and 3.4 g of N,N′-dicyclohexylcarbodiimide to give the desired compound.
    • Yield :
    5.0 g
    M.P. :
    201 - 202°C
    Rf¹ :
    0.26 Rf² : 0.55
    [α]D :
    +2.1° (c=0.5, DMF)
    (2) Boc-Pro-Arg(Mbs)-Gly-NH₂
  • The procedure of (2) in Example 1 was repeated using 20.8 g of Z-Arg(Mbs)-Gly-NH₂, 12.1 g of Boc-Pro-OSu and 4.3 ml of N-methylmorpholine to give the desired compound.
    • Yield :
    21.5 g
    M.P. :
    120 - 126°C
    Rf¹ :
    0.31 Rf² : 0.53
    [α]D :
    -26.5° (c=1, DMF)
    (3) Boc-Cys(MBzl)-Pro-Arg(Mbs)-Gly-NH₂
  • 3.7 g of Boc-Pro-Arg(Mbs)-Gly-NH₂ was subjected to 4N HCl-ethyl acetate treatment in the same manner as (3) in Example 1 to remove Boc.
  • In 30 ml of DMF was dissolved the obtained H-Pro-Arg(Mbs)-Gly-NH₂ hydrochloride, and to the solution were successively added under chilling with ice 0.7 ml of N-methylmorpholin, 2.1 g of Boc-Cys(MBzl)-OH, 0.85 g of 1-hydroxybenzotriazole and 1.4 g of N,N′-dicyclohexylcarbodiimide. After stirring for 18 hours at room temperature, the reaction mixture was filtered to remove N,N′-dicyclohexylurea, and then the filtrate was treated to distill off DMF.
  • In CHCl₃ was dissolved the residue, the resulting solution was then washed successively with a saturated aqueous sodium hydrogencarbonate solution, dilute hydrochloric acid saturated with sodium chloride and a saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate.
  • The solvent was distilled off, and ether was added to the resulting residue for crystallization. The crystals were collected by filtration to yield the desired compound.
    • Yield :
    3.2 g
    M.P. :
    104 - 107°C
    Rf¹ :
    0.44 Rf² : 0.63
    [α]D :
    -27.9° (c=0.5, DMF)
    (4) Z-pGlu-Asn-Cys(MBzl)-Pro-Arg(Mbs)-Gly-NH₂
  • To 10 ml of 2N HCl-acetic acid was added 1.8 g of Boc-Cys(MBzl)-Pro-Arg(Mbs)-Gly-NH₂. The mixture was allowed to stand for 30 min. at room temperature and then treated to distill off the solvent.
  • The residue was dried under reduced pressure, and then dissolved in 30 ml of DMF. To the resulting solutin were added under chilling with ice 0.25 ml of N-methylmorpholine, 0.9 g of Z-pGlu-Asn-OH, 0.38 g of 1-hydroxybenzotriazole and 0.5 g of N,N′-dicyclohexylcarbodiimide.
  • After stirring for 40 hours, the mixture was filtered to remove N,N′-dicyclohexylurea, and then the filtrate was treated to distill off DMF.
  • In 2-butanol-dichloromethane (5:1 v/v) was dissolved the residue. The solution was washed successively with a saturated aqueous sodium hydrogencarbonate solution, dilute hydrochloric acid saturated with sodium chloride, and a saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate.
  • The solvent was distilled off, and the residue was purified by silica gel chromatography using chloroform-methanol, and then crystallized by addition of ether and collected by filtration to give the desired compound.
    • Yield :
    0.85 g
    M.P. :
    131 - 135°C
    Rf¹ :
    0.19 Rf² : 0.44
    [α]D :
    -43.3° (c=0.6, DMF)
    (5) pGlu-Asn-Cys-Pro-Arg-Gly-NH₂ acetate
  • To a mixture of 4 ml of methanesulfonic acid, 0.25 ml of anisol and 0.2 ml of ethanedithiol was added 440 mg of z-pGlu-Asn-Cys(MBzl)-Pro-Arg(Mbs)-Gly-NH₂, and the mixture was stirred for 1 hour at room temperature. To the mixture was added ether, and the supernatant portion of the mixture was removed. The precipitate was dissolved in water. The solution was subjected to Dowex 1x2 (acetate type) treatment, and then was treated to distill off the water.
  • The residue was dissolved in 0.05 % trifluoroacetica cid, the solution was purified using a high-performance liquid chromatography at 15 ml/min. (flow rate), 2 to 12 % B) 20 min. linear gradient (mobile phase). The resulting solution was treated with Dowex 1x2 (acetate type) treatment and freeze-dried to give the desired compound.
    • Yield :
    28 mg
    Rf³ (including 1% ethanedithiol) :
    0.14
    [α]D :
    -92.8° (c=0.5, water)
    Example 3
  • Figure imgb0009
    • (1) Boc-D-Pro-Arg(Mbs)-Gly-NH₂
  • The procedure of (2) in Example 1 was repeated using 5.2 g of Z-Arg(Mbs)-Gly-NH², 3.1 g of Boc-D-Pro-OSu, and 2.2 ml of N-methylmorpholine to give the desired compound.
    • Yield :
    5.7 g
    M.P. :
    88 - 91°C
    Rf¹ :
    0.35 Rf² : 0.59
    [α]D :
    +8.7° (c=0.6, DMF)
    (2) Boc-Cys(Acm)-D-Pro-Arg(Mbs)-Gly-NH₂
  • The procedure of (3) in Example 1 was repeated using 2.5 g of Boc-D-Pro-Arg(Mbs)-Gly-NH₂, 1.3 g of Boc-Cys(Acm)-OH, 0.73 g of 1-hydroxybenzotriazole, 0.94 g of N,N′-dicyclohexylcarbodiimide and 1 ml of N-methylmorpholine to give the desired compound.
    • Yield :
    2.2 g
    M.P. :
    110 - 114°C
    Rf¹ :
    0.22 Rf² : 0.50
    [α]D :
    -21.9° (c=0.5, DMF)
    (3) Z-pGlu-Asn-Cys(Acm)-D-Pro-Arg(Mbs)-Gly-NH₂
  • The procedure of (4) in Example 1 was repeated using 2.0 g of Boc-Cys(Acm)-D-Pro-Arg(Mbs)-Gly-NH₂, 2.0 g of Z-pGlu-Asn-OH, 0.5 g of 1-hydroxybenzotriazole, 0.58 g of N,N′-dicyclohexylcarbodiimide and 0.5 ml of N-methylmorpholine to give the desired compound.
    • Yield :
    1.8 g
    M.P. :
    120 - 124°C
    Rf¹ :
    0.09 Rf² : 0.35
    [α]D :
    -23.3° (c=0.5, DMF)
    (4) Z-pGlu-Asn-Cys(Scm)-D-Pro-Arg(Mbs)-Gly-NH₂
  • The procedure of (5) in Example 1 was repeated using 1.0 g of Z-pGlu-Asn-Cys(Acm)-D-Pro-Arg(Mbs)-Gly-NH₂ and 0.15 ml of Cl-Scm to give the desired compound.
    • Yield :
    0.9 g
    M.P. :
    142 - 147°C
    Rf¹ :
    0.20 Rf² : 0.49
    [α]D :
    -40.8° (c=0.5, DMF)
    Figure imgb0010
       The procedure of (6) in Example 1 was repeated using 0.5 g of Z-pGlu-Asn-Cys(Scm-D-Pro-Arg(Mbs)-Gly-NH₂ and 0.15 g of cysteine hydrochloride to give the desired compound.
    Yield :
    0.46 g
    M.P. :
    162 - 165°C
    Rf² :
    0.07
    [α]D :
    -26.2° (c=0.5, DMF)
    Figure imgb0011
       120 mg of
    Figure imgb0012
    was subjected to methanesulfonic acid(MSA)-anisole treatment in the same manner as (7) in Example 1, and then purified by a high-performance liquid chromatograpy at 12 ml/min. (flow rate), 0 to 20% B) 20 min. linear gradient (mobile phase). The resulting solution was subjected to Dowex 1x2 (acetate type) treatment, freeze-dried to give the desired compound.
    Yield :
    53 mg
    Rf³ :
    0.10
    [α]D :
    -106.0° (c=0.5, water)
    Example 4
  •    H-Asn-Cys-Pro-Arg-OH acetate
    • (1) Boc-Pro-Arg(Mbs)-OBzl
  • To a solution of 14.2 g of H-Arg(Mbs)-OBzl hydrochloride in 200 ml of THF were added 3.3 ml of N-methylmorpholine and 9.4 g of Boc-Pro-OSu. The mixture was stirred for 18 hours at room temperature.
  • THF was distilled off, and the residue was dissolved in ethyl acetate. The solution was successively washed with an aqueous dilute hydrochloric acid, a saturated aqueous sodium hydrogencarbonate solution and water, and dried over anhydrous sodium sulfate.
  • The solvent was distilled off to give the desired compound as an oil.
    • Yield :
    18 g
    Rf¹ :
    0.69 Rf² : 0.86
    [α]D :
    -29.6° (c=0.5, DMF)
    (2) Boc-Cys(MBzℓ)-Pro-Arg(Mbs)-OBzl
  • To 15 ml of 4N HCl-ethyl acetate was added 3.7 g of Boc-Pro-Arg(Mbs)-OBzl. The mixture was allowed to stand for 30 minutes at room temperature, and the solvent was then removed by distillation. The residue was dried under reduced pressure, and then dissolved in 50 ml of DMF. To the solution were added under chilling with ice 1.4 ml of N-methylmorpholine, 2.2 g of Boc-Cys(MBzl)-OH, 0.95 g of 1-hydroxybenzotriazole and 1.3 g of N,N′-dicyclohexylcarbodiimide. After stirring 18 hours at room temperature, the reaction mixture was filtered to remove N,N′-dicyclohexylurea, and the filtrate was treated to distill off DMF.
  • The resulting residue was dissolved in 2-butanol-dichloromethane (5:1 v/v), and the solution was washed successively with a saturated aqueous sodium hydrogencarbonate solution, dilute hydrochloric acid saturated with sodium chloride and a saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate.
  • The solvent was distilled off, and the residue was purified by shilica gel chromatography using chloroformmethanol to yield the desired compound as an oil.
    • Yield :
    4 g
    Rf¹ :
    0.82 Rf² : 0.88
    [α]D :
    -25.0° (c=0.5, DMF)
    (3) Z-Asn-Cys(MBzl)-Pro-Arg(Mbs)-OBzl
  • To 5 ml of 4N HCl-ethyl acetate was added 1.7 g of Boc-Cys(MBzl)-Pro-Arg(Mbs)-OBzl. The mixture was allowed to stand for 30 min. at room temperature, and the solvent was removed. To the residue were added 2-butanoldichloromethane (5:1 v/v) and a saturated aqueous sodium hydrogencarbonate solution. The organic portion was taken out, and washed with a saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate.
  • The solvent was distilled off, and the residue was dissolved in 30 ml of DMF. To the resulting solution were added under chilling with ice 0.58 g of Z-Asn-OH, 0.34 g of 1-hydroxybenzotriazole and 0.45 g of N,N′-dicyclohexylcarbodiimide. After stirring 18 hours at room temperature, N,N′-dicyclohexylurea was removed from the mixture by filtration and DMF was distilled off from the filtrate.
  • The residue was dissolved in 2-butanol-dichloromethane (5:1 v/v), and the solution was washed successively with a saturated aqueous sodium hydrogencarbonate solution, dilute hydrochloric acid saturated with sodium chloride and a saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate.
  • After the solvent was distilled off, the residue was crystallized from ether, and the crystals were collected by filtration to yield the desired compound.
    • Yield :
    1.8 g
    M.P. :
    98 - 100°C
    Rf¹ :
    0.70 Rf² : 0.82
    [α]D :
    -29.2° (c=0.5, DMF)
    (4) H-Asn-Cys-Pro-Arg-OH acetate
  • To a mixture of 4 ml of methanesulfonic acid and 0.4 ml of anisol was added 100 mg of Z-Asn-Cys(MBzl)-Pro-Arg(Mbs)-OBzl. The mixture was stirred for 1.5 hour at room temperature and, after addition of ether, the supernatant portion of the solution was removed. The precipitate was dissolved in water. The solution was subjected to Dowex 1x2 (acetate type) treatment, and the water was distilled off.
  • The residue was dissolved in 0.05 % trifluoroacetic acid, and the solution was purified by high-performance liquid chromatography at 12 ml/min. (flow rate), 0 to 10% B) 20 min. linear gradient (mobile phase) and subjected to Dowex 1x2 (acetate type) treatment to freeze-dry and to obtain the desired compound.
    • Yield :
    47 mg
    Rf³ (including 1% ethanedithiol) :
    0.18
    [α]D :
    -54.6° (c=0.5, water)
       Examples of pharmacological tests showing the effectiveness of the peptides of the present invention are set forth below. Pharmacological Tests
  • The effect of peptides of the invention on memory consolidation was evaluated by conducting one-trial passive avoidance experiment using male Wistar rats in accordance with the method described by Burbach et al., Science, vol. 221, pp. 1310-1312, 1983. The apparatus was composed of an illuminated room and a dark room, and their floors were made of stainless-steel grid. The rats placed in the illuminated room could freely enter the dark room. Upon entering the dark room the rats received an electro-shock. Retention of passive avoidance behavior to the electro-shock was determined by the measurement of a response latent period, i.e. period required for the rat experienced the electro-shock to reenter the dark room from the time at which the rat was placed in the illuminated room after predetermined intervals.
    • (1) Examination on facilitation effect of memory consolidation
  • The rats were treated with the peptides of the invention obtained in the aforementioned Example 1 to 4 or a physiological saline solution by means of subcutaneous injection immediately after receiving the electro-shock (0.25 mA). Then 24 hours later, the retention of the memory of the elctro-shock was tested.
  • The rats administered with the physiological saline solution alone as a control group generally showed response latent period of approx. 50 seconds.
  • For the comparison, the above same tests were conducted with the following known peptides.
    • Comparison Compound 1 (known peptide):
  • Figure imgb0013
    • Comparison Compound 2 (known peptide):
  • Figure imgb0014
    For each group, 6 to 8 rats were tested. The response latent period was measured up to a maximum of 600 seconds.
  • The dose and the effect (the ratio of the response latent period of each group to that of the control groups, shown as %) of the peptides obtained in each example and the peptides of each comparison compound are set forth in Table 1. Table 1
    Group Dose (ng/kg) Effect (%)
    Example 1 1 267
    Example 2 1 295
    Example 3 1 253
    Example 4 1 316
    Comparison 1 10 313
    Comparison 2 10 249
    • 2) Examination on improvement effect of experimental retrograde amnesia by cycloheximide
  • The rats received an electro-shock (0.5 mA) after one hr. from the administration of the peptides of the invention or a physiological saline solution. Immediately after receiving the electro-shock, the rats were treated with 2.7 to 3.0 mg/kg of cycloheximide or the saline solution by subcutaneous injection. At 48 hours after the administration was made, memory retentions of the rats were tested. The rats administered with only the physiological saline solution showed the response latent period of approx. 300 seconds, and those rats of control group administered with a physiological saline and treated with cycloheximide alone showed the response latent period of approx. 50 seconds, which revealed retrograde amnesia.
  • The average response latent period of rats administered with each peptide of the invention and treated with cycloheximide were compared with that of the control group. Six to eight rats were used for each group to be tested. The response latent period was measured up to a maximum of 600 seconds.
  • The dose and the effect (the ratio of response latent period of each group to that of the control groups, shown as %) of the peptides obtained in each example and the peptides of each comparison example are set forth in Table 2. Table 2
    Group Dose (ng/kg) Effect (%)
    Example 1 1 302
    Example 2 1 633
    Example 3 1 380
    Example 4 1 320
    Comparison 1 10 583
    Comparison 2 100 503
  • As is readily apparent from the above experimental results, the peptides of the invention had the same effects as the known peptides at a dose of 1/10 to 1/100 to that of the known peptides and showed superior effect on the facilitation of memory consolidation as well as effect on improving retrograde amnesia.
  • Although the structures of the peptides of the invention have similar structures to those of the known peptides, they are slightly different from those of the known peptides which hold the significant influence on memory consolidation effect of a peptide. The fact that these slight defferences made the peptides of the invention produce remarkable effect indicates that it is impossible to make an estimation on memory consolidation effect of a peptide from its structure, and that the peptides of the invention have the uniqueness on their effects.
  • For example, although the peptide obtained in Example 1 possesses D-Arg in place of L-Arg of the peptide of Comparison Compound 1 and the rest of the structure of each peptide is identical, the peptide obtained in Example 1 showed the same effect to that of Comparison Compound 1 with only 1/10 of dose, which testifies the fact the the peptide obtained in Example 1 has much seperior effect to the known peptide.
    • Preparation Example 1 (Injection)
  • To 100 ml of a distilled water for injection were added 0.1 mg of the peptide obtained in Example 1 and 0.9 g of sodium chloride to prepare an aqueous solution whose pH was adjusted to 6.0 to 8.0 with sodium hydroxide. The solution was filtered under sterile condition, and the filtrate was filled up into 1 ml ampul. The ampul was fused to seal under sterile condition by heating to prepare an agent for injection.
    • Preparation Example 2 (Freeze-Dried Agent)
  • To 100 ml of a distilled water for injection were added 5 mg of the peptide obtained in Example 1 and 5 g of D-mannitol to prepare an aqueous solution of which pH was adjusted to 6.0 to 8.0 with a phosphate buffer. The solution was filtered under sterile condition and the filtrate was divided into a plurality of 1 ml vials. The divided portions were freeze-dried to prepare a freeze-dried agent for injection.
    • Preparation Example 3 (Collunarium)
  • To 100 ml of a physiological saline solution was added 10 mg of the peptide obtained in Example 1. The pH of the mixture was adjusted to 3.0 to 6.0 with a citric acid buffer to prepare a collinarium which contains 50 µg of the peptide of the invention in a dose of 0.5 ml.
    • Preparation Example 4 (Suppository)
  • To 98.5 g of hard fat (triglyceride of saturated fatty acid) was added 0.5 of egg york lecithin. The mixture was melted at temperature of 40 to 45°C and to the melted mixture was added under stirring a solution of 5 mg of the peptide (obtained in Example 1) in 1 g of Polyethylene glycol (PEG) 400. The resulting dispersion (1 g) was filled into the mold for suppository. The content was removed from the mold after being caked to prepare a suppository.

Claims (5)

  1. A peptide having the formula (I):
    Figure imgb0015
     functional derivatives thereof selected from derivatives of N-acyl groups of formula -NHCOR where R is an alkyl group having 1-5 carbon atoms; amide, monoalkyl- or dialkyl-substituted amides of formula -CONH₂, -CONHR or -CONR₂ where R is an alkyl group having from 1 to 6 atoms; and ester groups of formula -COOR¹ where R¹ is an alkyl group having from 1-18 carbon atoms,
    or a pharmaceutically-acceptable salt thereof.
  2. A peptide having the formula (II):



            pGlu-Asn-Cys-Pro-Arg-Gly   (II)



    functional derivatives thereof selected from derivatives of N-acyl groups of formula -NHCOR where R is an alkyl group having 1-5 carbon atoms; amide, monoalkyl- or dialkyl-substituted amides of formula -CONH₂, -CONHR or -CONR₂ where R is an alkyl group having from 1 to 6 atoms; and ester groups of formula -COOR¹ where R¹ is an alkyl group having from 1-18 carbon atoms,
    or a pharmaceutically-acceptable salt thereof.
  3. A peptide having the formula (III):
    Figure imgb0016
     functional derivatives thereof selected from derivatives of N-acyl groups of formula -NHCOR where R is an alkyl group having 1-5 carbon atoms; amide, monoalkyl- or dialkyl-substituted amides of formula -CONH₂, -CONHR or -CONR₂ where R is an alkyl group having from 1 to 6 atoms; and ester groups of formula -COOR¹ where R¹ is an alkyl group having from 1-18 carbon atoms,
    or a pharmaceutically-acceptable salt thereof.
  4. A peptide having the formula (IV):



            Asn-Cys-Pro-Arg   (IV)



    functional derivatives thereof selected from derivatives of N-acyl groups of formula -NHCOR where R is an alkyl group having 1-5 carbon atoms; amide, monoalkyl- or dialkyl-substituted amides of formula -CONH₂, -CONHR or -CONR₂ where R is an alkyl group having from 1 to 6 atoms; and ester groups of formula -COOR¹ where R¹ is an alkyl group having from 1-18 carbon atoms,
    or a pharmaceutically-acceptable salt thereof.
  5. An anti-dementia agent containing an effective dose of a peptide having a formula selected from the group consisting of the following formulae (I), (II), (III) and (IV):
    Figure imgb0017

            pGlu-Asn-Cys-Pro-Arg-Gly   (II)

    Figure imgb0018

            Asn-Cys-Pro-Arg   (IV)



    functional derivatives thereof selected from derivatives of N-acyl groups of formula -NHCOR where R is an alkyl group having 1-5 carbon atoms; amide, monoalkyl- or dialkyl-substituted amides of formula -CONH₂, -CONHR or -CONR₂ where R is an alkyl group having from 1 to 6 atoms; and ester groups of formula -COOR¹ where R¹ is an alkyl group having from 1-18 carbon atoms,
    or a pharmaceutically-acceptable salt thereof and, a pharmaceutically acceptable carrier or a diluent.
EP89308221A 1988-08-12 1989-08-14 Novel peptide and anti-dementia agent Expired - Lifetime EP0354819B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AT89308221T ATE104314T1 (en) 1988-08-12 1989-08-14 PEPTIDE AND AGAINST DEMENTIA.

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP63201358A JP2654673B2 (en) 1988-08-12 1988-08-12 Peptides and anti-dementia agents
JP63201359A JP2542241B2 (en) 1988-08-12 1988-08-12 peptide
JP201358/88 1988-08-12
JP201359/88 1988-08-12

Publications (3)

Publication Number Publication Date
EP0354819A2 EP0354819A2 (en) 1990-02-14
EP0354819A3 EP0354819A3 (en) 1990-12-19
EP0354819B1 true EP0354819B1 (en) 1994-04-13

Family

ID=26512745

Family Applications (1)

Application Number Title Priority Date Filing Date
EP89308221A Expired - Lifetime EP0354819B1 (en) 1988-08-12 1989-08-14 Novel peptide and anti-dementia agent

Country Status (7)

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US (1) US5180712A (en)
EP (1) EP0354819B1 (en)
KR (1) KR0144005B1 (en)
AU (1) AU626574B2 (en)
CA (1) CA1328949C (en)
DE (1) DE68914544T2 (en)
DK (1) DK398089A (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0620230A1 (en) * 1989-04-15 1994-10-19 Nippon Chemiphar Co., Ltd. Peptides and antidementia agents containing the same
WO1997039026A1 (en) 1996-04-15 1997-10-23 Kabushiki Kaisha Yakult Honsha Novel peptides and nootropic agent
US6193993B1 (en) * 1998-03-03 2001-02-27 Eisai Co., Ltd. Suppository containing an antidementia medicament

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IE56021B1 (en) * 1982-10-13 1991-03-27 Akzo Nv Peptides

Also Published As

Publication number Publication date
DK398089D0 (en) 1989-08-14
CA1328949C (en) 1994-04-26
DK398089A (en) 1990-02-13
AU3990889A (en) 1990-02-15
US5180712A (en) 1993-01-19
KR0144005B1 (en) 1998-07-01
EP0354819A3 (en) 1990-12-19
AU626574B2 (en) 1992-08-06
DE68914544T2 (en) 1994-08-25
DE68914544D1 (en) 1994-05-19
EP0354819A2 (en) 1990-02-14
KR900003201A (en) 1990-03-26

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